Biomedical subjects

Tsuyoshi Miyazaki

Publications and source records attributed to Tsuyoshi Miyazaki.

10 recordsLinked to original sources

PubMed2004

Src kinase activity is essential for osteoclast function.

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.

Adenoviridae↗

PubMed2004

Investigation of unsaturated water flow in a deep vadose zone by applying quasi-partition law to 222Rn.

This paper describes an application of the quasi-partition law of radon-222 (222Rn) to a deep unsaturated vadose zone in a field. 222Rn is a very useful tracer to investigate not only saturated but also unsaturated water flows in vadose zones. Concentrations of 222Rn in water in unsaturated soils are, based on the quasi-partition law, lower than those in saturated soils. However, no study has previously applied quasi-partition law of 222Rn to analyze saturated and unsaturated water flows in vadose zones. In this study, we observed the applicability of existing theoretical equations of quasi-partition law of 222Rn experimentally. After confirming the validity of the theoretical equation, we applied this equation to estimate the unsaturated water flow in an actual vadose zone. By comparing the estimated degree of saturation from 222Rn measurement with that from a neutron moisture meter measurement, we obtained a beautiful agreement between them.

Environmental Monitoring↗

PubMed2004

[TNF-alpha].

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Animals↗

PubMed2003

Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3-only Bcl-2 family member Bim.

Osteoclasts (OCs) undergo rapid apoptosis without trophic factors, such as macrophage colony-stimulating factor (M-CSF). Their apoptosis was associated with a rapid and sustained increase in the pro-apoptotic BH3-only Bcl-2 family member Bim. This was caused by the reduced ubiquitylation and proteasomal degradation of Bim that is mediated by c-Cbl. Although the number of OCs was increased in the skeletal tissues of bim-/- mice, the mice exhibited mild osteosclerosis due to reduced bone resorption. OCs differentiated from bone marrow cells of bim-/- animals showed a marked prolongation of survival in the absence of M-CSF, compared with bim+/+ OCs, but the bone-resorbing activity of bim-/- OCs was significantly reduced. Overexpression of a degradation-resistant lysine-free Bim mutant in bim-/- cells abrogated the anti-apoptotic effect of M-CSF, while wild-type Bim did not. These results demonstrate that ubiquitylation-dependent regulation of Bim levels is critical for controlling apoptosis and activation of OCs.

Animals↗

PubMed2003

Tyrosine phosphatase epsilon is a positive regulator of osteoclast function in vitro and in vivo.

Protein tyrosine phosphorylation is a major regulator of bone metabolism. Tyrosine phosphatases participate in regulating phosphorylation, but roles of specific phosphatases in bone metabolism are largely unknown. We demonstrate that young (<12 weeks) female mice lacking tyrosine phosphatase epsilon (PTPepsilon) exhibit increased trabecular bone mass due to cell-specific defects in osteoclast function. These defects are manifested in vivo as reduced association of osteoclasts with bone and as reduced serum concentration of C-terminal collagen telopeptides, specific products of osteoclast-mediated bone degradation. Osteoclast-like cells are generated readily from PTPepsilon-deficient bone-marrow precursors. However, cultures of these cells contain few mature, polarized cells and perform poorly in bone resorption assays in vitro. Podosomes, structures by which osteoclasts adhere to matrix, are disorganized and tend to form large clusters in these cells, suggesting that lack of PTPepsilon adversely affects podosomal arrangement in the final stages of osteoclast polarization. The gender and age specificities of the bone phenotype suggest that it is modulated by hormonal status, despite normal serum levels of estrogen and progesterone in affected mice. Stimulation of bone resorption by RANKL and, surprisingly, Src activity and Pyk2 phosphorylation are normal in PTPepsilon-deficient osteoclasts, indicating that loss of PTPepsilon does not cause widespread disruption of these signaling pathways. These results establish PTPepsilon as a phosphatase required for optimal structure, subcellular organization, and function of osteoclasts in vivo and in vitro.

Animals↗

PubMed2003

Regulation of cytochrome c oxidase activity by c-Src in osteoclasts.

The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane-associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria.

Animals↗

PubMed2003

Suppression of arthritic bone destruction by adenovirus-mediated dominant-negative Ras gene transfer to synoviocytes and osteoclasts.

OBJECTIVE: To determine the role of Ras-mediated signaling pathways in synovial cell activation and bone destruction in arthritic joints. METHODS: The E11 rheumatoid synovial cell line and primary synovial fibroblast-like cells (SFCs) from patients with rheumatoid arthritis (RA) were gene-transferred by replication-deficient adenovirus vector carrying the dominant-negative mutant of the ras gene (AxRasDN). The effects of RasDN overexpression on cellular proliferation, interleukin-1 (IL-1)-induced activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK], p38, c-Jun N-terminal kinase [JNK]), and IL-6 production by synovial cells were analyzed. The in vivo effects of Ras inhibition on synovial cell activation and arthritic bone destruction were analyzed by injection of AxRasDN into ankle joints of rats with adjuvant arthritis. RESULTS: AxRasDN markedly reduced the proliferation of RA SFCs. IL-1, a proinflammatory cytokine involved in RA pathology, induced activation of ERK, p38, and JNK in the cells. Adenovirus vector-mediated RasDN overexpression suppressed ERK activation, but not p38 or JNK activation, in SFCs. IL-6 is also an important proinflammatory cytokine, and RasDN inhibited IL-1-induced production of IL-6 by RA SFCs at both the transcriptional and protein levels. Injection of AxRasDN into ankle joints of rats with adjuvant arthritis ameliorated inflammation and suppressed bone destruction in the affected joints. CONCLUSION: Ras-mediated signaling pathways are involved in the activation of RA SFCs and the destruction of bone in arthritic joints, suggesting that inhibition of Ras signaling can be a novel approach for RA treatment that targets both synovial cell activation and bone destruction in the RA joint.

Adenoviridae↗

PubMed2002

IL-1 regulates cytoskeletal organization in osteoclasts via TNF receptor-associated factor 6/c-Src complex.

Targeted disruption of either c-Src or TNFR-associated factor 6 (TRAF6) in mice causes osteoclast dysfunction and an osteopetrotic phenotype, suggesting that both molecules play important roles in osteoclastic bone resorption. We previously demonstrated that IL-1 induces actin ring formation and osteoclast activation. In this study, we examined the relationship between IL-1/TRAF6-dependent and c-Src-mediated pathways in the activation of osteoclast-like cells (prefusion cells (pOCs); multinucleated cells) formed in the murine coculture system. In normal pOCs, IL-1 induces actin ring formation and tyrosine phosphorylation of p130(Cas), a known substrate of c-Src. However, in Src-deficient pOCs, p130(Cas) was not tyrosine phosphorylated following IL-1 treatment. In normal pOCs treated with IL-1, anti-TRAF6 Abs coprecipitate p130(Cas), protein tyrosine kinase 2, and c-Src. In Src-deficient pOCs, this molecular complex was not detected, suggesting that c-Src is required for formation of the TRAF6, p130(Cas), and protein tyrosine kinase 2 complex. Moreover, an immunocytochemical analysis revealed that in osteoclast-like multinucleated cells, IL-1 induced redistribution of TRAF6 to actin ring structures formed at the cell periphery, where TRAF6 also colocalized with c-Src. Taken together, these data suggest that IL-1 signals feed into the tyrosine kinase pathways through a TRAF6-Src molecular complex, which regulates the cytoskeletal reorganization essential for osteoclast activation.

Actins↗

PubMed2002

Joint proprioception before and after total knee arthroplasty.

To investigate the effects of total knee arthroplasty on joint proprioception, the absolute angular error of the knee in 38 consecutive patients before and after total knee arthroplasty for osteoarthritis and in 23 age-matched control subjects were examined. Varus and valgus laxity of the knee and muscle strengths of the thigh were measured using appropriate instruments. There were no significant differences in absolute angular error before and after total knee arthroplasty, independent of retaining or substituting the posterior cruciate ligament. The absolute angular error of the knee with a normal appearing anterior cruciate ligament was larger than that with a missing anterior cruciate ligament before total knee arthroplasty and decreased significantly after surgery. The absolute angular error correlated with the varus and valgus laxity of the knee, but did not correlate with the strength of thigh muscles after total knee arthroplasty. These results suggest that deficiency of the anterior cruciate ligament may not adversely affect proprioception in severe knee osteoarthritis. In addition, proper ligament balance may partly contribute to better proprioception after total knee arthroplasty.

Aged↗

PubMed2002

Possible involvement of IkappaB kinase 2 and MKK7 in osteoclastogenesis induced by receptor activator of nuclear factor kappaB ligand.

Recent studies have revealed the essential role of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) in osteoclast differentiation and activation. Adenovirus vector could efficiently transduce genes into RAW264.7 cells, which differentiate into osteoclast-like multinucleated cells in the presence of RANKL. The role of NF-kappaB and c-jun N-terminal kinase (JNK) activation in RANKL-induced osteoclast differentiation was investigated using an adenovirus vector carrying the dominant negative 1kappaB kinase 2 gene (AxIKK2DN) or dominant negative MKK7 gene (AxMKK7DN). IKK2DN and MKK7DN overexpression in RAW cells specifically suppressed the NF-kappaB activation and JNK activation in response to RANKL, respectively, without affecting other signaling pathways. Either inhibition of NF-kappaB or JNK pathways dose-dependently inhibited osteoclast formation induced by RANKL. These results suggest that both NF-kappaB and JNK activation are independently required for osteoclast differentiation.

Adenoviridae↗